This invention relates to an analytical technique useful in predicting bioavailability of metal ions and amino acids from metal proteinate complexes for use in animal diet supplementation.
The importance of the trace elements copper, iron, manganese and zinc in animal nutrition has long been recognized. The beneficial effect of iron on blood formation was recognized as early as the 17th century. In 1928, Hart, et al. provided the first conclusive evidence that copper was required for recovery from anemia in rats. Zinc was shown to be required for normal growth and health in rats in 1934 and is now considered to be essential for the health of plants, animals and humans. The essential role of manganese for the health of animals and human has been recognized since as early as 1936.
For proper health, trace elements must be provided in the diet in sufficient quantities and in a form which can be utilized by the animal. The degree to which an ingested substances is absorbed in a form that is utilizable is defined as xe2x80x9cbioavailabilityxe2x80x9d. The bioavailability of nutrients for animals is summarized in a comprehensive monograph edited by Ammerman, Baker and Lewis published in 1995.
Many commercial products have been developed as additives to enhance the bioavailability of the trace elements from animal feed. The beneficial effects of these products are attributed to the association of the metal with an organic molecule, usually called a ligand. This association or bonding results in the increased availability of the metal for utilization by animals, i.e. increased bioavailability. The increased bioavailability of the trace elements in these products is a result of increased solubility, greater stability in the gut, enhanced absorption into circulation and/or improved metabolic utilization.
Different types of products that contain a trace element associated with an organic ligand are commercially available. These can be classified in different groups based on the nature of the ligand used in manufacturing the product. In one class of products, amino acids are used as the ligands that form complexes or chelates with the metal. Examples of these products are described in U.S. Pat. Nos. 3,941,818, 3,950,372, 4,067,994, 4,900,651, 4,948,594, 4,956,188, 5,061,815, 5,278,329, 5,583,243, 4,863,898 and 6,166,071. A second group of feed additives include the metal salts of short chain carboxylic acids such as propionic acid (see U.S. Pat. Nos. 5,591,878, 5,707,679, 5,795,615 and 5,846,581). A third group of trace element additives is classified by the American Feed Control Officials as Metal Proteinate and defined as xe2x80x9cthe product resulting from the chelation of a soluble salt with amino acids and/or partially hydrolyzed proteinxe2x80x9d. Examples of these products are described in U.S. Pat. Nos. 3,440,054, 3,463,858, 3,775,132, 3,969,540, 4,020,158, 4,076,803, 4,103,003, 4,172,072 and 5,698,724.
Determination of the composition of the metal amino acid complexes and the metal salts of short chain carboxylic acids usually involves standard analytical methods for the determination of metals and amino acids or carboxylic acids. On the other hand, metal proteinates consist of complex mixtures of metal and a protein source. The determination of the precise composition of these proteinates is not possible because of the differences in the source and degree of hydrolysis of the protein. In some products the protein is reported to be completely hydrolyzed to the constituent amino acids by acid and enzymatic hydrolysis. Other products contain partially hydrolyzed or even un-hydrolyzed proteins. Although some methods have been reported for evaluating metal proteinates based on solubility or free metal ions activity, there is no method available for the systematic and comprehensive analysis of these products to determine if the metal and the amino acid really are bioavailable to the livestock being fed.
To evaluate the quality of commercially available metal proteinates, it is important to determine both the degree of association of the metal with the protein molecules in these products and the nature of the protein moiety. For example, a composition that has metal ions associated with folded, non-hydrolyzed protein polymer may have the same empirical formula as metal-free amino acid complex, but the bioavailability may be dramatically different.
Unfortunately, there are no current methods available for the determination of the amount of metal associated with the protein polymer molecules, or free amino acids of different molecular weights, that are usually present in these products. And, if such a technique were available it would be a useful tool in addressing effectiveness of trace element mixes to provide bioavailable supplementation of both amino acids and trace elements.
Accordingly, an objective of this invention is to provide a simple method for the analysis of metal proteinates that allows for the determination of the amount of protein present in the different molecular weight fractions and the amount of metal associated with each of these fractions. In this way, one can deduce the lowest molecular weight fractions that are associated, and from this deduce effectiveness.
Another object of the invention is to provide molecular sieve ultrafiltration data for soluble trace mineral/protein complexes, and to use the profiles of data to predict bioavailability.
An even further objective is to provide a simple and effective test which can be used to compare and predict effectiveness of trace mineral products to achieve their commercial purposes.
A still further objective is to provide a test method that is run only on the soluble portion of a trace mineral product, recognizing that the insoluble portions are not truly bioavailable.
Another objective is to provide an assay which is truly and objectively predictive of how commercially available metal proteinate complexes will perform in field trials and daily use with livestock.
The method and manner of accomplishing these and other objectives of the present invention(s) will be apparent from the detailed description of the invention(s) which follows.